Gene therapy techniques utilizing prodrug activation genes have shown substantial therapeutic promise for cancer treatment. Prodrug activation-based cancer gene therapy is an attractive strategy to sensitize tumor cells to cytotoxic drugs, since the observed chemosensitization can extend beyond the cells that express the prodrug-activation gene to include surrounding tumor cells, allowing for an effective chemotherapeutic response even if only a minor fraction of tumor cells is transduced with the therapeutic gene. This proposal focuses on a novel prodrug activation cancer gene therapy strategy developed during the past project period. This strategy is based on the combination of a cytochrome P450 gene with a cancer chemotherapeutic prodrug, such as cyclophosphamide, which is activated via a P450-catalyzed monooxygenase reaction. The cancer therapeutic potential of P450 gene transfer derives, in part, from the striking cytotoxic enhancement that is associated with intratumoral, as compared to hepatic, prodrug activation. This approach is unique among current prodrug activation strategies, insofar as it utilizes mammalian genes in combination with proven and tested chemotherapeutic prodrugs currently employed in cancer treatment, rather than novel prodrugs whose ultimate therapeutic efficacy is uncertain. The proposed studies will test a series of hypotheses developed to further improve the high activity and the selectivity of antitumor responses associated with P450 gene therapy. The specific objectives of this project are: 1) to enhance the selectivity and the potency of intratumoral P450-catalyzed prodrug activation; 2) to increase the chemosensitivity' of the target tumor cell; 3) to improve the tumor targeting specificity for P450 gene delivery; and 4) to integrate the most promising of these advances into preclinical models of in vivo gene delivery and cancer prodrug chemotherapy. Together, these studies will establish a rational basis for enhancing intratumoral activation of established cancer chemotherapeutic drugs in a manner that decreases systemic toxic responses and improves therapeutic effects, and will thereby advance the development and implementation of P450-based gene therapy for cancer treatment.